Foxp3

The flow cytometry procedure for identification and quantification of circulating inflammatory and immune cells was based on our previous report [34 (link)]. Prior to sacrificing the animals, peripheral blood mononuclear cells (PBMCs) were obtained from the tail vein using a 27# needle. PBMCs (1.0 × 10⁶ cells) were triple-stained with FITC-anti-CD3 (BioLegend), PE-anti-CD8a (BD Bioscience, San Jose, CA, USA), and PE-Cy™5 anti-CD4 (BD Bioscience, San Jose, CA, USA). To identify CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Tregs), PBMCs were triple-stained with Alexa Fluor® 488-anti-CD25 (BioLegend, San Diego, CA, USA), PE-anti-Foxp3 (BioLegend, San Diego, CA, USA), and PE-Cy™5 anti-CD4 (BD bioscience, San Jose, CA, USA) according to the manufacturer's protocol for the Foxp3 Fix/Perm buffer set. The numbers of CD3⁺CD4⁺ helper T cells, CD3⁺CD8⁺ cytotoxic T cells and CD4⁺CD25⁺Foxp3⁺ Tregs were analyzed using flow cytometry (FC500, Beckman Coulter, Brea, CA, USA).
Additionally, the numbers of inflammatory cells in circulation [i.e., CD11b/c, LyG6, vascular cell adhesion molecule (VCAM)-1], in ascites [macrophage migratory inhibitor factor (MIF), CD14, CD11b/c, LyG6, CD68/CD80, CD68/CD163], and in ABL (CD11b/c, MIF, Ly6G) were assessed using the flow cytometric method.

The frequency of CD3+ T cells and CD4+FOXP3+ T-regulatory cells was analyzed in all samples by a CyAn flow Cytometer (Beckman Coulter, High Wycombe, Buckinghamshire, UK). Expression of CD3, CD4, and forkhead box 3 (FOXP3) (BioLegend, San Diego, California, USA) in combination with Programmed cell death protein 1 (PD1), Lymphocyte-activation gene 3 (LAG3), and T-cell immunoglobulin and mucin-domain containing-3 (BioLegend) exhaustion markers were investigated on frozen peripheral blood mononuclear cell samples. To assess the activation status of these T cells, 0.5 × 10⁶ peripheral-blood mononuclear cells were treated with 20 ng/mL phorbol 12-myristate 13-acetate (Sigma, St Louis, Missouri, USA) and 500 ng/mL ionomycin (Sigma) in vitro for 48 hours. Supernatants were harvested and the cytokine profile was analyzed by bead-array flow cytometry (BioLegend).